The present invention is directed to a new phenotype of stem cells which contain a fibroblast growth factor receptor (FGFR) on the cell surface thereof and further have a phenotype indicative of a primitive state. The present invention is further directed to subpopulations thereof having a phenotype indicative of endothelial or stromal cells.
The ability of tissues and organs to develop, remodel, regenerate, and repair depends on the existence of stem cells (also known as progenitor cells) that, upon division, form more differentiated progeny. Stem cells have been found in the epidermis, the intestinal epithelium, and the hematopoietic system. There is mostly indirect evidence of stem cells in mesenchymal tissues. In vivo and in vitro studies have provided evidence of osteogenic precursor cells in bone marrow and other stromal cell preparations. However, the identity of cells in these tissues and their relationship to cells with classical stem cell characteristics have yet to be established.
Endothelial cells are part of the normal bone marrow stroma. Long-term cultures of human bone marrow contain a complex mix of stromal cells including adipocytes, fibroblasts, endothelial cells, macrophages, and smooth muscle cells. Endothelial cells and hematopoietic cells are thought to be derived from the common progenitor cells, hemangioblasts.
Cell surface molecules on various types of cells, and particularly hematopoietic cells, are given a cluster of differentiation (CD) designation in which each CD molecule designation describes a surface molecule (marker) identifiable by a cluster of monoclonal antibodies that display the same cellular reactivity. CD designations are assigned at regularly held international workshops on human leukocyte differentiation antigens. For example, the CD19 marker is specific to B cells, and the CD33 marker is specific to myeloid cells. At the present time, it is not known how many of the markers associated with differentiated cells are also present on stem cells. One marker which has been indicated as being present on stem cells is CD34. However, this marker is also found on a significant number of lineage-committed progenitors. Other markers which are known or thought to be present primarily on stem cells, i.e., xe2x80x9cprimitivexe2x80x9d markers, include AC133 (Yin et al, 1997; Buhring et al, 1999), Thy-1 (Murray et al, 1995) and c-kit (D""Arena et al, 1998).
It is known that a small number of circulating CD34+ hematopoietic stem cells are present in peripheral blood. As the major source of CD34+ hematopoietic stem cells in the adult is the bone marrow, the purpose of this small, circulating CD34+ cell population is unknown. One explanation is that the bone marrow is xe2x80x9cleakyxe2x80x9d, and the stem cells escape, circulate and return to the marrow. A second possibility is that the function of these circulating stem cells is to seed sites, such as the liver and the spleen, which can function as additional sites of hematopoiesis in a crisis.
The human CD34+ hematopoietic population isolated from bone marrow, cord blood, and peripheral blood is a heterogeneous population that contains hematopoietic stem cells. Recent evidence indicates that circulating CD34+ cells also contain endothelial stem cells, which may also circulate (Asahara et al, 1997; Nieda et al, 1997; Shi et al, 1998; Lin et al, 1998). Asahara et al (1997) have shown that CD34+ cells isolated from the peripheral blood can be incorporated into the endothelium of ischaemic blood vessels of recipient animals. Purified umbilical cord blood CD34+ cells also give rise to von Willebrand factor-expressing endothelial cells in vitro, providing additional evidence for a circulating progenitor endothelial cell (Nieda et al, 1997). In addition, bone marrow derived CD34+ cells also contain a transplantable stromal stem cell (Prockop, 1997; Pereira et al, 1998).
Recently, convincing evidence has been presented (Goan et al, 1997) that human CD34+ progenitor cells from peripheral blood or cord blood that were transplanted into NOD/SCID immunodeficient mice gave rise to human stromal cells. The human stromal cells expressed the endothelial cell-specific vascular endothelial growth factor (VEGF) receptor-2 (KDR) and von Willebrand factor, indicating that they were of endothelial origin. There is also recent evidence that infusion of whole bone marrow cells into recipient mice results in fibroblasts of donor origin in a number of non-hematopoietic tissues (Prockop, 1997; Pereira, 1998), indicating that stromal progenitor cells reside in the bone marrow. As CD34 has been shown to be expressed by bone marrow stromal precursor cells (Simmons et al, 1991), it is possible that these stromal progenitors reside in the bone marrow within the CD34+ progenitor population.
The CD34+ progenitor population is, therefore, a heterogeneous fraction that may include precursor cells of the hematopoietic, endothelial, and stromal/fibroblast lineages. In addition, pluripotent mesenchymal stem cells capable of differentiating into cells of the osteogenic, chondrogenic, tendonogenic, adipogenic and myogenic lineages have been shown to reside within the bone marrow microenvironment (Majumdar et al, 1998). There is recent literature indicating that circulating endothelial progenitor/stem cells exist, and that stromal stem cells in marrow serve as a source for continual renewal of cells in a number of non-hematopoietic tissues. A common embryological precursor that gives rise to both hematopoietic and endothelial cells has recently been identified (Suda et al, 1997; Choi et al, 1998; Caprioli et al, 1998).
Recent evidence has also shown that embryonic stem (ES) cells can give rise to endothelial cells (Hirashima et al, 1999).
Fibroblast growth factors (FGFs) can synergize with other factors to stimulate hematopoietic progenitor cell proliferation (Wilson et al, 1991; Quito et al, 1996; Allouche, 1995; Yuen et al, 1998, U.S. Pat. No. 5,612,211; U.S. Pat. No. 5,817,773). It has also been shown that basic FGF (FGF-2) acts to antagonize cytokines that induce differentiation (Burger et al, 1994). In addition, low amounts of FGF-2, on the order of 10-100 pg/ml, induce a more primitive phenotype in human K562 leukemic cells.
It would be very useful to be able to isolate stem cells which are progenitors of endothelial and/or stromal cells. The more primitive the stem cell, the more useful it is in bone marrow transplantation. Furthermore, endothelial stem cells and stromal stem cells, or a stem cell which is a progenitor of both, would find many utilities in repairing damaged vasculature and in treating other conditions where endothelial or stromal cells need to be replenished.
It is an object of the present invention to overcome the deficiencies in the prior art.
It is another object of the present invention to isolate a new phenotype of stem cells.
It is a further object of the present invention to identify and isolate stem cells for use in stem cell transplantation.
It is yet another object of the present invention to identify and isolate endothelial and/or stromal stem cells.
A small population of cells having a xe2x80x9cprimitive phenotypexe2x80x9d, such as CD34+ or CD34xe2x88x92linxe2x88x92, has been isolated which express cell surface receptors for fibroblast growth factor (FGF). The population of cells bearing FGF receptors (FGFR) are designated as FGFR+. The FGFR+ primitive phenotype cell population has several unique properties:
(1) The CD34+FGFR+ cells are predominantly present in the region of the fluorescence-activated cell sorter profile having low forward scatter (FSC) and low side scatter (SSC). Thus, the majority of the cells of the population are very small and of low granularity. These small cells are located in the FSC/SSC region of the fluorescence-activated cell sorter profile that is normally not analyzed, as this area contains many of the dead and apoptotic cells. Interestingly, this region has recently been shown to be the site of a mesenchymal stem cell population (Zohar et al, 1997). The CD34xe2x88x92linxe2x88x92FGFR+ cells have FSC/SSC properties that are similar to those of the CD34+FGFR+ cells. This CD34xe2x88x92linxe2x88x92FGFR+ population also includes significant numbers of cells with higher FSC properties.
(2) The CD34+FGFR+ cells are deeply dormant, which is characteristic of a stem cell population. They do not proliferate in culture until 30-60 days after isolation.
The FGFR+ primitive phenotype cell population is a unique stem cell population that is a precursor cell for endothelium and/or hematopoiesis and/or stroma. The FGFR+ primitive phenotype cell population, obtained either from general circulation, the bone marrow, cord blood or embryonic cells, is capable of forming endothelial, blood and stromal cells, depending on the need at the time.